A proprietary water soluble extract from the roots of North American ginseng (Panax quinquefolium), CVT-E002, is commercially available as COLD-FX™. This extract differs from other Asian or American ginseng products in the content of polysaccharides and ginsenosides, primarily consisting of poly-furanosyl-pyranosyl-saccharides. Batch-to-batch quality of the product is certified by ChemBioPrint™ technology, which assures its chemical as well as pharmacological consistency. This proprietary natural extract is known to have immunomodulatory effects (Wang et al. 2001, 2004). CVT-E002 enhances the proliferation of mouse spleen cells, and increases production of interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF)-α and nitric oxide (NO) from peritoneal macrophages in vitro. Administration of CVT-E002 to mice increased serum immunoglobulin G (IgG) antibody levels (Wang et al., 2001) and daily dosing of CVT-E002 to mice with viral-induced leukemia increased the proportions of macrophages and NK cells in the bone marrow and spleen while reducing the leukemic cell numbers (Miller, 2006). In a recent study on human peripheral blood mononuclear cells (PBMC) cultured with live influenza virus, CVT-E002 was effective in enhancing the production of IL-2 and interferon γ (IFNγ) (Jing et al., submitted). IL-2 and IFNγ are major T and NK cell cytokines and are associated with virus-specific adaptive immune responses. In a clinical study, daily low dose supplementation of COLD-FX™ to healthy adults increased the proportion of NK cells in plasma (Predy et al., 2006).
Being the first line of defense against microbial pathogens, both macrophages and NK cells are important components of innate immunity. These cells act immediately to limit proliferation and spread of infectious agents through release of antimicrobial agents such as cytokines, interferons and chemokines and by their phagocytic or cytolytic activities.
Since pre-clinical studies suggested potential use of CVT-E002 for the prophylaxis of virus-related upper respiratory infections, a clinical trial involving 198 institutionalized seniors was conducted. This study demonstrated that daily administration of CVT-E002 for 4 months during an influenza season reduced relative risk of acute respiratory illness due to influenza and respiratory syncytial virus by up to 89% (McElhaney et al., 2004). Another study also showed CVT-E002 significantly reduced the recurrence of respiratory infections in 323 healthy middle-aged adults (Predy et al., 2005). CVT-E002 treatment also reduced the severity and duration of symptoms related to upper respiratory tract infections in healthy adults. In a randomized double-blind, placebo controlled trial of 43 community-dwelling adults aged 65 or older, daily ingestion of CVT-E002 reduced the relative risk and duration of respiratory symptoms by 48% and 55%, respectively. Daily CVT-E002 administration was shown to be a safe, natural therapeutic means of prevention of acute respiratory illness in healthy seniors.
The mammalian immune system has evolved multiple, layered and interactive defensive systems to protect against infections, which have been broadly divided into innate immunity and adaptive immunity. Innate immunity is the first line of defense against microbial pathogens and acts almost immediately to limit early proliferation and spread of infectious agents through activation of phagocytic and antigen-presenting cells, such as dendritic cells and macrophages, and initiation of inflammatory responses through the release of a variety of cytokines, chemokines and anti-microbial factors, such as interferons and defensins. Innate immunity is evolutionarily ancient and for many years its study was largely ignored by immunologists as relatively non-specific. For the most part, humans are protected against infection by the innate immune system. If infectious organisms penetrate innate immune defenses, the innate defenses facilitate and guide the generation of adaptive immune responses that are directed against highly specific determinants that are uniquely expressed by the invading pathogen. These responses are dependent on rearrangement of specific antigen-receptor genes in B-cells and T-cells and result in production of high-affinity antigen-specific antibodies (humoral immunity) and T-cells or cell-mediated immunity. Antibodies facilitate removal, destruction or neutralization of extracellular pathogens and their toxins. T-cell-mediated immune responses help eliminate or control intracellular pathogens. In contrast to innate immune responses, adaptive immune responses have the hallmark of specific immune memory.
Previous studies have attempted to determine how the host innate immune system detects infection and how it discriminates between self and pathogens or infectious non-self. The discovery and characterization of Toll-like receptors (TLRs) have provided great insight into innate immune recognition and established a key role of the innate immune system in host defense against infection (Akira et al., 2006; Hargreaves and Medzhitov, 2005; Kawai and Akira, 2006; Philpott and Girardin, 2004; Seth et al., 2006). TLRs are key molecules in innate and adaptive immunity. The innate immune system uses multiple families of germline-encoded pattern recognition receptors (PRRs) to detect infection and trigger a variety of antimicrobial defense mechanisms (Janeway and Medzhitov, 1998). These PRRs are evolutionarily highly conserved among species from plants and fruit flies to mammals. The strategy of innate immune recognition is based on the detection of highly conserved and essential structures present in many types of microorganisms and absent from host cells (Janeway, 1992; Janeway and Medzhitov, 1999). Since the targets of innate immune recognition are conserved molecular patterns, they are called pathogen-associated molecular patterns (PAMPs). PAMPS have important features that make them ideal targets for innate immune sensing. PAMPs are produced only by microorganisms and not by host cells. This is the basis for discrimination of self and infectious non-self. PAMPs are conserved between microorganisms of a given class, allowing a limited number of PRRs to detect the presence of a large class of invading pathogens. For example, a pattern in LPS allows a single PRR to detect the presence of any Gram-negative bacteria. PAMPs are essential for microbial survival and any mutation or loss of PAMPs is either lethal for the organism or greatly reduces their adaptive fitness. These new insights into innate immune recognition are revolutionizing the understanding of immune defense, pathogenesis, and treatment and prevention of infectious diseases.
TLRs represent one family of PRRs that are evolutionarily conserved transmembrane receptors that detect PAMPs and function as signaling receptors. TLRs were discovered in Drosophila where they play a role in development of the fruit flies ventral/dorsal orientation (Stein et al., 1991). When this gene was mutated, the flies that developed were found to be “toll” which is German slang for crazy or “far out.” Further, flies with mutation of Tolls were found to be highly susceptible to fungal infections (Lemaitre at al., 1996). To date, 11 TLRs have been identified in mammals, each sensing a different set of microbial stimuli and activating distinct signaling pathways and transcription factors that drive specific responses against the pathogens (Kawai and Akira, 2005). TLRs are type I integral membrane glycoproteins characterized by extracellular domains containing various numbers of leucine-rich-repeat (LRR) motifs, a transmembrane domain and a cytoplasmic signaling domain homologous to that of the interleukin-1 receptor (IL-1R), termed the Toll/IL-1R homology (UR) domain (O'Neill, 2006). The LRR domains are composed of 19-25 tandem LRR motifs, each of which is 24-29 amino acids in length.
TLR4, the first mammalian TLR discovered, proved to be the long sought receptor for Gram-negative bacterial lipopolysaccharide (LPS) (Medzhitov et al., 1997; Poltorak et al., 1998). TLR2 recognizes peptidoglycan, in addition to the lipoproteins and lipopeptides of Gram-positive bacteria and mycoplasma (Takeda et al., 2003; Takeuchi et al., 1999). TLR2 can form heterodimers with TLR1 or TLR6 to discriminate between diacyl and triacyl lipopeptides, respectively (Takeda et al., 2003). Further, TLR2 in collaboration with the non-TLR receptor dectin-1 mediates the response to zymosan, found in the yeast cell-wall (Gantner et al., 2003). TLR5 recognizes flagellin, a protein component of bacterial flagella (Hayashi et al., 2001). TLR11, a close relative of TLR5, was found to be abundantly expressed in the urogenital tract of mice and was associated with protection against uropathogenic bacteria (Zhang et al., 2004), and was recently shown to recognize profilin-like protein from the protozoan parasite Toxoplasma gondii (Yarovinsky et al., 2005). TLR3, 7, 8 and 9 recognize nucleic acids and are not expressed on the cell surface, but are exclusively expressed in endosomal compartments (Latz et al, 2004; Matsumoto et al., 2003). TLR3 is involved in recognition of double-stranded RNA (dsRNA) generated during viral infection (Alexopoulou et al., 2001), whereas closely related TLR 7 and 8 recognize viral single stranded (ss)RNA rich in guanosine or uridine (Diebold at al., 2004; Heil at al, 2004) and synthetic imidazoquinoline-like molecules, imiquimod and resiquimod (R-848) (Hemmi at al., 2002; Jurk et al., 2002). TLR9 mediates the recognition of bacterial and viral unmethylated CpG DNA motifs (Hemmi et al., 2000) and was recently also shown to recognize non-DNA pathogenic components, such as hemozoin from malarial parasites (Coban et al., 2005). TLR10 plays a role in the pathogen-mediated inflammation pathway, pathogen recognition and activation of innate immunity, but the TLR10 ligand is presently unknown.
TLRs can also be divided into six major subfamilies based on sequence similarity (Roach et al., 2005), each recognizing related PAMPS. The subfamily consisting of TLR1, TLR2 and TLR6 recognizes lipopeptides, TLR3 recognizes dsRNA, TLR4 LPS, TLR5 flagellin, and the TLR9 subfamily that includes highly related TLR7 and TLR8 recognize nucleic acids. Importantly, the subcellular localization of TLRs correlates with the nature of their ligands, rather then sequence similarity (Hargreaves and Medzhitov, 2005). TLR1, 2, 4, 5, 6 and 10 are present on the surface plasma membrane where they are involved in the pathogen mediated inflammation pathway and/or recognize bacterial and viral components, while antiviral TLRs, TLR3, 7, 8, and 9 are expressed in intracellular endosomes. Since nucleic acids recognized by antiviral TLRs are also found in vertebrates, their location in endosomes limits their reactivity to self nucleic acids (Barton et al., 2006). TLR11 is present on the cell surface and is a receptor for uropathogenic bacteria and protozoan parasites.
Signaling by TLRs is complex and has been reviewed elsewhere (Akira and Takeda, 2004; O'Neill, 2006). Briefly, all TLRs with the exception of TLR3 signal through the adaptor molecule myeloid differentiation factor 88 (MyD88), a cytoplasmic protein containing a TIR domain and a death domain. Ultimately, NF-κB and MAPKs are activated downstream of TRAF6 leading to production of proinflammatory cytokines and chemokines, such as TNF-α, IL-6, IL-1β and IL-12. In addition to MyD88, TLR3 and TLR4 signal through TRIF, another TIR-containing adaptor that is required for production of type I interferons and type I interferon-dependent genes.
TLRs are expressed on a variety of immune and non-immune cells. Murine macrophages express TLR1-9, reflecting their importance in the initiation of proinflammatory responses. Plasmacytoid DCs (pDCs) that produce large amounts of type I interferons during viral infections express TLR7 and 9. All conventional DCs in the mouse express TLR1, 2, 4, 6, 8 and 9, while TLR3 is confined to the CD8+ and CD4− CD8− DC subset (Iwasaki and Medzhitov, 2004). In humans, TLR9 expression is restricted to pDCs and B-cells (Bauer et al., 2001; Krug et al., 2001).
There is great interest in understanding expression of TLRs on mucosal epithelial cells (ECs) that serve as the first line of defense against most infections. In our recent studies (Yao X-D et al., 2007), we have concentrated on understanding expression and regulation of TLRs on ECs in the genital tract of mice and humans. Laser capture microdissection (LCM) was used to show that the estrous cycle in female mice profoundly influences expression of TLRs in the vaginal epithelium. mRNA expression of essentially all TLRs except TLR11 were significantly increased during diestrus and especially following treatment with the long acting progestin Depo-Provera (Yao X-D et al., manuscript submitted). These findings contribute to our understanding of innate immune defense against sexually-transmitted infections, and enhance the quality of female reproductive health.
Mucosal delivery of TLR ligands, including CpG oligodeoxynucleotides (ODN which is a ligand for TLR9), dsRNA, and flagellin, can induce an innate anti-viral effect that can protect mice against intravaginal (IVAG) challenge with HSV-2 (Ashkar and Rosenthal, 2002). Studies have showed that intranasal administration of purified envelope glycoprotein (gB) from HSV-2 plus CpG ODN as an adjuvant induced strong gB-specific IgA and IgG in the vaginal tract (persisting throughout the estrous cycle) as well as systemic and genital gB-specific CTL, and protected against lethal IVAG HSV-2 infection (Gallichan et al., 2001). Subsequently, it was shown that intranasal immunization with inactivated gp120-depleted HIV-1 plus CpG ODN induced anti-HIV IgA in the genital tract and HIV-specific T-cell-mediated immune responses, including production of IFNγ and β-chemokines (Dumais et al., 2002). Further, mice immunized intranasally with HIV-1 plus CpG induced CD8+ T-cells in the genital tract, providing cross-clade protection against IVAG challenge with recombinant vaccinia viruses expressing HIV-1 gag from different clades (Jiang et al., 2005). More recently, although the genital tract has been considered to be a poor immune inductive site, especially following immunization with non-replicating antigens, intravaginal (IVAG) immunization of female mice with recombinant subunit HSV-2 gB plus CpG induced higher levels of gB-specific IgG and IgA antibodies in serum and vaginal washes versus mice immunized with antigen alone and mice immunized with gB plus CpG were better protected against vaginal infection with HSV-2 (Kwant and Rosenthal, 2004). Thus, it is possible to induce protective immune responses following IVAG immunization with a non-replicating subunit protein antigen provided an appropriate mucosal adjuvant is used.
Recent studies have shown that PAMPs including CpG DNA, dsRNA, and LPS were capable of inhibiting herpes simplex virus type 2 (HSV-2) and vesicular stomatitis virus (VSV) in vitro (Ashkar et al., 2003 & 2004). A single dose of CpG ODN delivered transmucosally to the vaginal mucosa, in the absence of any viral antigen, protected against genital infection with lethal doses of HSV-2. This protection was mediated by the innate immune system, since it occurred in knockout mice lacking B and T cells. Local IVAG delivery of CpG ODN resulted in rapid proliferation and thickening of the vaginal epithelium and induction of a TLR-9-dependent antiviral state that did not block virus entry but inhibited viral replication in vaginal epithelial cells (Ashkar et al., 2003). Mucosal delivery of dsRNA, the ligand for TLR3, protected against genital HSV-2 infection without the local or systemic inflammation seen with CpG ODN (Ashkar et al., 2004). Therefore, local delivery of TLR3 ligand may be a safer means of protecting against genital viral infection.
TLRs induce a range of responses depending on the cell type in which they are activated (Ashkar and Rosenthal, 2002; Iwasaki and Medzhitov, 2004). For example, treatment of DCs with CpG DNA that acts through TLR9 activates the DCs to mature, including upregulation of MHC class II and costimulatory molecules, as well as production of proinflammatory cytokines, chemokines and enhancement of antigen presentation. Similarly, treatment of B-cells with CpG induces their activation and proliferation, secretion of antibody as well as IL-6 and IL-10 and the B-cells become resistant to apoptosis. Activation of immune cells via CpG DNA induces a Th1-dominated response.
The mechanisms by which PRRs mediate host defense against pathogens are the focus of intense research. Due to their ability to enhance innate immune responses, there is a need for novel strategies to use ligands, synthetic agonists or antagonists of PRRs (i.e., “innate immunologicals”) as stand alone agents to provide protection or treatment against infection with intracellular bacteria, parasites and viruses. Further, activation of innate immune system through PRRs using their respective ligands or agonists represents a strategy to enhance immune responses against specific pathogens, making agents which signal via PRRs potential vaccine adjuvants.
There is a need for a natural, herbal fraction or composition which specifically activates the innate and adaptive immune responses to treat associated conditions such as allergies, asthma, viral and microbial infections, and cancer without causing deleterious side effects or discomfort. The types of immune responses are well known. Th1 responses are characterized by the generation of killer T cells and certain antibodies in response to intracellular pathogens and intracellular defects such as cancers. Th2 responses fight extracellular pathogens. Allergic reactions occur in response to environmental substances (i.e., allergens), and are the result of specific Th2 responses. Th2 responses are characterized by the generation of other specific types of antibodies and are typical of allergic reactions, in which an allergen is mistaken for a pathogen on a mucosal surface and triggers an immune response resulting in symptoms such as watery eyes, airway inflammation and contraction of airway muscle cells in the lungs. TLR activation induces antigen-presenting cells to produce cytokines that favor Th1-type immune responses, thereby preventing or reducing the development of deleterious Th2 responses due to exposure to allergens.
Allergies are specifically characterized by excessive activation of white blood cells called mast cells and basophils by IgE, resulting in an extreme inflammatory response. When an allergy-prone person is initially exposed to an allergen, large amounts of the corresponding, specific IgE antibody are made. The IgE molecules attach to the surface of mast cells (in tissue) or basophils (in the circulation). Mast cells are found in the lungs, skin, tongue, and linings of the nose and intestinal tract. When an IgE antibody on a mast cell or basophil encounters its specific allergen, the IgE antibody signals the mast cell or basophil to release chemicals such as histamine, heparin, and substances that activate blood platelets and attract secondary cells such as eosinophils and neutrophils. The activated mast cell or basophil also synthesizes new mediators, including prostaglandins and leukotrienes. These chemical mediators cause the symptoms associated with allergies, including wheezing, sneezing, runny eyes and itching. Common allergic reactions include eczema, hives, hay fever, asthma, food allergies, and reactions to the venom of stinging insects such as wasps and bees.
An asthma exacerbation is a serious deterioration in the lung function of a patient often resulting in hospitalization and even death. Asthma occurs when the main air passages of the lungs, the bronchial tubes, become inflamed. The muscles of the bronchial walls tighten, and cells in the lungs produce extra mucus further narrowing the airways, causing minor wheezing to severe difficulty in breathing. Asthma is often triggered by a respiratory viral infection, such as the common cold, but other irritants such as cigarette smoke, dust mites, animal dander, plant pollen, air pollution, deodorants and perfume can make asthma symptoms more frequent, severe, and uncontrollable. Other asthma triggers include, exercise, cold air, and emotional stress. The majority of asthma exacerbations are precipitated by common airway virus infections. In children, being atopic and having a virus infection are both major risk factors for being admitted to a hospital for a wheezing illness. While the clinical importance of asthma attacks and specifically viral exacerbation of asthma is clear, the reasons why patients with asthma become so ill after common cold viruses remains poorly understood.
Normally, viral infections cause an influx of neutrophils into the airways with a large mononuclear cell component of predominantly CD8+ T-cells. However, it has become apparent that viral infections can produce a range of inflammatory responses, including airway eosinophilia, depending on the pre-existing condition of the host. In atopic individuals, experimental rhinovirus infection increases the recruitment of eosinophils to the airways after antigen challenge and causes increased airway reactivity compared to non-allergic individuals. After intranasal infection with rhinovirus, biopsies of the lower airways of asthmatic individuals contain increased eosinophils, which persist even into convalescence. In patients with asthma, the presence of airway eosinophils during periods of exacerbations has been well established. The finding of eosinophils in airway during asthma exacerbation becomes somewhat paradoxical, considering that these exacerbations are often triggered by viral infection. While the association of eosinophils and their degranulation products in the airways has been described during virus infection in patients with asthma, whether eosinophils are active in response to the virus and how this activation might occur is unknown.
For an asthma exacerbation to occur, the current understanding suggests that effector cells (i.e. eosinophils, mast cells, basophils, neutrophils) may be activated. FIG. 1 illustrates a model of virus-induced eosinophil mediator release in the airway which results in airway hyperreactivity via dysfunction of the neural control of airway smooth muscle. Virus or virus antigen is presented to memory T-cells. Activated T-cells (CD4) release an unknown soluble degranulation factor, likely a cytokine such as GM-CSF. These T-cells may also express cell surface ligands, for example, ICAM-1. Eosinophils respond to the soluble mediator, cell surface ligands, or combination thereof with release of various eosinophil mediators (i.e. eosinophil major basic protein, eosinophil peroxidase, RANTES).
In asthmatics, virus-induced eosinophil mediator release in the airways correlates with the development of asthma exacerbation. For the eosinophil to be involved in the development of virus-induced asthma exacerbations, it must respond to the virus either indirectly via another cell or directly. This process would represent virus-induced eosinophil mediator release.
Western physicians have been reluctant to prescribe herbal medicines due to lack of scientific research of their preventative and therapeutic properties. However, herbal medicines do not require the lengthy development time and high costs normally encountered with synthetic drugs. Further, they are readily available and offer the subject a more comfortable and affordable alternative with minimal side effects compared to prescription medication or vaccines.